The art is generally cognizant of the basic technique of fusing mouse myeloma cells to spleen cells from immunized mice to obtain a culturable, continuous cell line making homogeneous or "monoclonal" antibodies. See, for example Kohler and Milstein, Nature 256, 495-497 (1975). However, particular difficulties and unique requirements are frequently encountered in attempts to apply this general knowledge, and even if hybridomas are successfully produced, there is no assurance that one will be obtained that produces an antibody having a desired specificity.
In the production of monoclonal antibodies and the hybridomas making them, a convenient experimental animal, such as a mouse, is exposed to the antigen against which an antibody is desired. Typically, some of the antigen is injected into the animal, and its immune system is allowed to respond to it. This process may be repeated until the animal's immune system is presumed to be producing antibodies to the antigen, as well as such other antibodies as the animal may be producing without regard to the injections of the antigen. The animal is then killed, and antibody-producing cells from it are isolated. Typically spleen cells from the animal are employed.
A large number of such spleen cells are then fused with myeloma cells of the same species to obtain hybrid cells that will reproduce without the self-limiting growth characteristics of most non-tumor cells. The fused cells are then cultured as cell lines of genetically identical, antibody producing cells. However, there is no assurance that the antibody produced by any particular cell line is an antibody to the original antigen or that the antibody will be specific to the antigen.
In order to select from among the many hybridoma cell lines thus produced for a particular cell line that produces a desired antibody, it is necessary to screen the cell lines. Typically this is done by testing the antibody produced by each cell line against the original antigen or a purified form thereof. The cell lines that are found by this means to produce a desired antibody are then preserved, and the remainder are discarded.
Many antigens are complex and may be species specific in at least certain respects. Thus, if mouse cells of a given type are used as the antigen in the process reviewed above, the antibody produced by a resulting hybridoma may be specific to any of many antigenic features of such cells or combinations of such features. If, for example, endothelial cells are employed, the antibody produced may be to an antigenic feature typical of endothelial cells in general, of mouse endothelial cells, of mouse cells regardless of their origin, and so forth. There is no assurance whatsoever that the antibody will be specific against endothelial cells of other species. Even within a given species, it has been shown that antibodies specific to endothelial cells taken from a given organ or tissue may not react in the same way to like cells from other organs or tissues. See J. Joseph, et al., Endothelial Cell Identification and Culture Methods, D. Thilo, ed., Karger, Basel (in press).
Certain enzymes, especially when in situ on cells, may also constitute antigens of sufficient complexity that a monoclonal antibody produced against the antigen has an unpredictable specificity. Thus, enzymes operating on a like substrate but produced by different species may differ sufficiently in themselves and certainly in the way they are presented by the cells of each species that a monoclonal antibody developed against the enzyme as produced or situated in the mouse, for example, may not react with the corresponding enzyme as produced or situated in bovine or human cells. A monoclonal antibody to such an enzyme may recognize the enzyme only in the context of aspects of a cell that are species specific instead of recognizing the parts that relate solely to the substrate acted on by the enzyme. And, as mentioned above, parts of the enzyme itself may be species specific. For reasons of efficiency and flexibility, it is desirable to be able to produce monoclonal antibodies usable in assays and similar work that are specific to an enzyme without being excessively specific to the species source of the cell presenting the enzyme.
It should be emphasized again that the unpredictable nature of hybrid cell preparation generally does not allow one to extrapolate from one antigen or cell system to another to predict precise outcomes of the application of conventional hybridization techniques. The successful production of an antibody of effective cross-species specificity produced by a hybridoma generated in response to inoculation with antigen from a given species is even more incapable of prediction.
Angiotensin-converting enzymes (hereinafter "ACE") are produced by many species. It is unknown if ACE is the same in all species or differs from species to species. In any event, there is no way to predict the ability of an antibody to react to the ACE of different species in situ on endothelial cells. However, ACE as produced by each such species, functions in known ways to convert the decapeptide angiotensin I to an octapeptide angiotensin II. See Ryan, et al., U.S. Pat. No. 4,115,374.
Detecting the presence and levels of ACE itself has medical utility, as is discussed in Ryan, et al. Furthermore, the endothelial cells of species producing ACE may be distinguished from other cells of those organisms in that the endothelial cells interact with the ACE produced by the organism. The ACE becomes bound to the surface of the endothelial cells. Thus, an antibody specific to ACE becomes an effective way to distinguish endothelial cells. This is desirable as a tool in the operation of fluorescence actuated cell sorters and like assay and separation techniques and equipment. The antibody to ACE may be directly labeled in conventional ways for detection by radiological, fluorescent, enzyme reaction, or other means. Alternatively, the antibody to ACE may be itself unlabeled but be subjected to reaction with another, labeled antibody. Thus, if the original antibody to ACE was produced by a mouse, commercially available preparations of labeled goat anti-mouse or rabbit anti-mouse antibodies could be employed. The presence of such labeled antibodies may then be detected by conventional means to indirectly reveal the presence of the antibody to ACE.
In the past, production of anti-ACE antibodies has been possible by conventional inoculation of rabbits or other animals and the processing of serum later extracted from the inoculated animal. The attempted production of a monoclonal antibody to ACE has not been reported, much less the production of such an antibody having effective cross-species specificity.